Dry solid medium for storage and analysis of genetic material

ABSTRACT

The present invention provides for device for obtaining and storing genetic material. The device has a support and a head portion. The head portion is composed of a solid matrix for sorbing genetic material and a preserving mechanism for the protection of genetic material from degradation. Additionally, the present invention provides for a method of storing a sample of genetic material onto the swab device. In the preferred embodiment, the present invention utilizes polyester material as the solid matrix.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation-in-part of U.S. Patentapplication Ser. No. 09/335,656, filed Jun. 18, 1999, which is acontinuation of U.S. patent application Ser. No. 08/979,833, filed Nov.26, 1997, now U.S. Pat. No. 5,976,572, which is a continuation of U.S.patent application Ser. No. 08/480,135, filed Jun. 7, 1995, now U.S.Pat. No. 5,756,126, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/159,104, filed Nov. 30, 1993, now U.S. Pat. No.5,496,562, which is a continuation of abandoned U.S. patent applicationSer. No. 07/671,859, filed May 29, 1991, the disclosures of which areincorporated herein by reference. The reader is also advised of U.S.Ser. No. 08/320,041, filed Oct. 7, 1994, which is anothercontinuation-in-part of U.S. Ser. No. 08/159,104.

FIELD OF THE INVENTION

[0002] The field of the present invention pertains to a dry solid mediumand method for collection of genetic material in a form suitable forstorage and/or subsequent analysis. Specifically, the present inventionprovides for a sampling device and method for collecting geneticmaterial through the use of the dry solid medium. The invention furtherprovides for analysis of stored genetic material using methods that aresuited for automated analyzing systems

BACKGROUND

[0003] Blood containing genetic material to be analyzed has typicallybeen transported from the place of removal from a human or animal to theplace of analysis as purified genetic material, liquid whole blood,frozen whole blood or whole blood dried onto paper. All of the methodshave disadvantages. Transport of genetic material in blood as dried,purified genetic material is most desirable, but it requires a highstandard of technical assistance to be available at the place of removalfrom the human or animal. When technical assistance is not available atthe place of removal, whole blood or other unpurified samples areusually sent to a central facility where the genetic material ispurified.

[0004] Transport of liquid whole blood often involves the need forsterility of collection. Under some circumstances, this is extremelyinconvenient, for example, where the sample is a heel-prick taken froman infant. The transport of liquid whole blood or frozen blood alsodemands temperature control and an appropriate transport system otherthan the regular postal system. This is true even before consideringconcerns about hygiene. In addition, problems with pathogens associatedwith whole blood, such as the HIV virus, generally rule out thetransport of any potentially infectious liquid or frozen sample exceptunder proper and expensive supervision.

[0005] Blood dried on filter paper is a proven alternative to the aboveprocedures and it has been shown that genetic material can be extractedand isolated form dried whole blood spots in a form and in sufficientquantities for use in DNA analysis. McCabe, E. R. B., et al., “DNAMicroextraction From Blood Spots on Filter Paper Blotters: PotentialScreening Applications to Newborn Screening,” Hum. Genet. 75:213-216(1987). But, this procedure still suffers from a number ofdisadvantages. For example, typically, there has been no deliberate andrapid destruction of blood associated pathogens. This creates apotential hazard for blood handling personnel. In addition, usually,there has not been deliberate inhibition of the processes that degradethe genetic material other than occurring by desiccation. However, slowdesiccation, or a small degree of re-hydration under conditions ofrelatively high humidity, will allow the growth of DNA or RNA destroyingmicroflora. Moreover, even in the presence of a bacteriostatic agent ofthe type that does not denature proteins, there are conditions thatpermit enzymatic-autolytic breakdown of the genetic material and somenonenzymatic breakdown of the genetic material. (Enzymatic-autolyticbreakdown refers to the process whereby dying or damaged tissues, ofeither human, animal or parasite cells, activate enzymes that degradetheir own components). Furthermore, there is typically considerabledifficulty desorbing very high molecular weight DNA or RNA from paper,if this is required. Surface adsorption effects can cause losses ofgenetic material that may cause the preferential loss of the leastdegraded, i.e. the most desired class of DNA or RNA molecules.

[0006] Thus, there is a need for a safe, convenient and minimally laborintensive means for storage of a genetic material to be analyzed that iscontained in a liquid sample.

[0007] However, even if a sample of genetic material is collected in asafe, convenient and reliable form for storage and subsequent analysis,there are also logistic problems, which arise when there are manydifferent types of analysis to be performed on a collected sample. Forexample, polymerase chain reaction (PCR) analysis requires a differentprimer-pair for each specific analysis to be performed. Obviously, theproblems tend to further increase when multiple samples are submittedfor analysis.

[0008] Present methods for in-situ-processing based on the use ofoligonucleotide primers, for example, PCR, rely on the stored geneticmaterial being heated and cooled in reaction mixtures that have primersadded to them at the time of beginning temperature cycling. In manytypes of analysis of genetic material, it is the primers that determinethe particular specificity of a reaction. Because there is usually aprimer pair for each conceivable type of analysis, there are anextremely large number of possible primers (for example, all thesequences within the genes of humans, animals and all other livingorganisms including the pathogens of humans and animals). Thus, in anycentralized facility that receives multiple samples for analysis ofgenetic material using, for example, oligonucleotide primers, thelogistic problems can be immense.

[0009] Automation of analysis of genetic material allows for increasednumbers of samples to be processed more efficiently. However, automationof the analysis of genetic material still requires the automated systemto have completely separate delivery devices for each different set ofprimers. Otherwise, the cross-contamination that may occur will beimpossible to control.

[0010] Thus, in general, if reactions like PCR are to be carried out ata centralized location using automated systems and using presently knownmethods, the range of different sequences which can be analyzed at onetime will be restricted by the physical problems of keeping a cleandelivery system of pipettes, etc., or by the molecular problems inherentin using mixtures of diverse primers. Clearly, circumvention of thisrestriction would be advantageous for analyzing multiple samples ofgenetic material for different genetic sequences.

[0011] Finally, samples can be taken from various surfaces and areas totest samples for particular substances such as antigens or geneticmaterial. Currently, common methods can involve utilizing a swab orswab-like device. Generally, once the sample is obtained the sample istransferred to another medium to perform any subsequent analysis. Inmost cases, the swab is capable of being used on any surface, including,but not limited to skin, wounds, flesh, body orifices such as the mouth,and any other living or nonliving surface. Most swabs used and known tothose of skill in the art have the basic components of a narrow rodsupport in combination with a collecting medium positioned and attachedadjacent to the support.

SUMMARY OF THE INVENTION

[0012] The present invention provides a safe, convenient and minimallylabor intensive apparatus and method for storage of a genetic materialthat is contained in a liquid medium. The present invention specificallyprovides for a sampling device and method for collecting geneticmaterial through the use of a dry solid medium. The invention furtherprovides for storage of genetic material in such a way as to allow forsimplified analysis of one or more samples of genetic material usingautomated systems.

[0013] The invention provides a dry solid medium for storage of a sampleof genetic material. The dry solid medium of the invention is composedof a solid matrix and a composition which when applied to the dry solidmedium protects against degradation of genetic material stored on thedry solid medium. The dry solid medium further provides for inactivationof microorganisms, including those that may be pathogenic to humans.

[0014] The composition of the dry solid medium includes a weak base, achelating agent, and anionic detergent and optionally, uric acid or aurate salt.

[0015] According to the invention, genetic material stored on the drysolid medium may be analyzed using methods known in the art, forexample, polymerized chain reaction (PCR), ligase chain reaction (LCR),reverse transcriptase initiated PCR, DNA or RNA hybridization techniquesincluding restriction fragment length polymorphism (RFLP) and othertechniques using genetic or DNA or RNA probes, genomic sequencing,enzymic assays, affinity labeling, methods of detection using labels orantibodies and other similar methods.

[0016] Genetic material stored on the dry solid medium of the inventionmay be analyzed in situ or after removal from the dry solid medium.

[0017] Another embodiment of the invention provides a dry solid mediumfor storage of a sample of genetic material and includes a componentthat is functional in the subsequent analysis to be performed on thestored sample of genetic material. According to the invention,components for subsequent analysis which may be included with a sampleof genetic material include, for example, nucleotide sequences such asPCR primers, target sequence stabilizers, genetic probes, primers forgenetic sequencing or sets of oligonucleotide substrates for LCRanalysis.

[0018] The invention also provides methods for using the dry solid mediaof the invention. In one embodiment, the invention provides a method forstoring a sample of genetic material on the dry solid for storing asample of genetic material on the dry solid medium in a substantiallynon-degraded form.

[0019] The invention further provides a method for storing a sample ofgenetic material and subsequently analyzing the stored sample of geneticmaterial. According to this embodiment of the invention, the storedgenetic material is preferably washed to remove any proteins orhemoglobin associated with a sample of genetic material or to remove anycomponent of the composition of the dry solid medium which may inhibitsubsequent analysis of the genetic material. The invention provides forthe stored genetic material to be washed using either an aqueous or anon-aqueous washing system.

[0020] The aqueous and non-aqueous washing systems used to wash a sampleof genetic material prior to analyzing the genetic material provide forremoving protein, hemoglobin or components of the composition of the drysolid medium without substantially affecting the stored genetic materialor any component included on the dry solid medium for subsequentanalysis of the genetic material. In addition, the aqueous andnon-aqueous wash systems of the invention may be used to wash geneticmaterial that is stored, for example, on a solid matrix which does nothave sorbed to it the DNA protecting composition of the invention.

[0021] The non-aqueous wash system of the invention includes contactinga sample of genetic material stored on the dry solid medium of theinvention with a herein-described single-phase phenol solution. Thesingle-phase phenol solution is then removed from the dry solid mediumcontaining a sample of genetic material and the dry solid mediumcontaining a sample of genetic material is then contacted with anaqueous alcohol wash solution. The aqueous alcohol wash solution isremoved and the dry solid medium containing the sample of geneticmaterial may further be washed with an aqueous ionic solution. Thewashed sample of genetic material may then be analyzed by methodsrecited above.

[0022] A sample of genetic material stored on a dry solid media of theinvention may also be analyzed using standard enzymic tests used, forexample, to detect phenylketonuria (PKU) or galactosemia afterneutralizing the effects of protein denaturing components of the drysolid medium using, for example, a converter solution.

[0023] The dry solid medium and the dry solid medium includingcomponents for subsequent analysis is particularly useful for analysisof a genetic sample using automated systems.

[0024] A further embodiment of the present invention is the use of thedry solid medium as a swab device used to obtain and gather geneticmaterial samples. Additionally, the present invention provides for amethod of collecting genetic material samples utilizing the swab device.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Other advantages of the present invention will be readilyappreciated, as the same becomes better understood by reference to thefollowing detailed description when considered in connection with theaccompanying drawings wherein:

[0026]FIG. 1, Photograph of ethidium bromide strained gel of PCRamplified DNA from blood samples stored on a dry solid medium of theinvention.

[0027]FIG. 2, Photograph of ethidium bromide stained gel of PCRamplified DNA from blood samples stored on a dry solid medium of theinvention which included oligonucleotide primers for PCR analysis.

[0028]FIG. 3, Photograph of ethidium bromide stained gel of Eco R1digestion of DNA from human blood stored on the dry solid medium of theinvention.

[0029]FIG. 4, Photograph of ethidium bromide stained gel of genomic DNAmaterial. Lane 1 is a 1 kb marker, lane 2 is “traditional” genomic DNAmaterial, and the remaining 12 lanes are samples amplified from PET-FTAswabs.

[0030]FIG. 5, Multiplex PCR profile from gDNA isolated using anFTA-coated PET filter membrane swab wherein 10 μl of eluted gDNA is usedper reaction.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention provides a dry solid medium for collectionof genetic material in a form suitable for storage and subsequentanalysis. The invention further provides methods for subsequentlyanalyzing the collected genetic material. The dry solid medium andmethods of use, disclosed herein, provide a safe, convenient andreliable means for storing samples of genetic material in a manner thatprovides reliable accuracy of analytical results. Moreover, theinvention provides for enhanced convenience and efficient when multiplesamples are analyzed using automated systems, for example, at acentralized analyzing facility.

[0032] In several places throughout the present specification guidanceis provided through lists of examples. The inventors wish to make clearthat in each instance, the recited lists serve only as representationgroups. It is not meant, however, that the lists are exclusive.

[0033] As used herein, the phrase “genetic material” (GM) means eitheror both deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Accordingto the invention, a sample of GM is collected on the dry solid medium byremoving the sample from a source and applying the sample to theherein-described dry solid medium. Methods for removing a sample ofgenetic material form a source are known in the art. For example, asample of genetic material in blood may be removed from a human oranimal source by venipuncture and the sample then applied to the drysolid medium of the invention.

[0034] As used herein a “sample of genetic material” or “sample of GM”includes a liquid having dissolved, suspended, mixed or otherwisecontained therein, either or both DNA or RNA, cells which contain eitheror both DNA or RNA or cell components which contain either or both DNAor RNA. Once the sample of GM is applied to the dry solid medium, theliquid tends to evaporate (evaporation may be enhanced by a warm airdryer) leaving the DNA and/or RNA entrained to the dry solid medium in adry from. The GM entrained to the dry solid medium in “dry form” may bepurified DNA and/or RNA, semipurified DNA and/or RNA or DNA and/or RNAremaining in cells.

[0035] The sample of GM, which is applied to the dry solid medium, maybe derived from any source. This includes, for example,physiological/pathological body liquids (e.g., secretions, excretions,exudates and transudates) or cell suspensions (e.g., blood, lymph,synovial fluid, semen, saliva containing buccal cells, skin scrapings,hair root cells, etc.) of humans and animals; physiological/pathologicalliquids or cell suspensions of plants; liquid products, extracts orsuspensions of bacteria, fungi, plasmids, viruses etc.; liquid products,protozoa, spirochetes, etc.; liquid extracts or homogenates of human oranimal body tissues (e.g., bone, liver, kidney, etc.); media from DNA orRNA synthesis; mixtures of chemically or biochemically synthesized DNAor RNA; and any other source in which DNA and/or RNA is or can be in aliquid medium. Preferably, the liquid containing the GM evaporates afterapplying the sample to the dry solid medium leaving GM in dry from priorto subsequent analysis.

[0036] The dry solid medium of the invention provides for storage and/orsubsequent analysis of the stored sample of GM. The dry solid medium iscomposed of a solid matrix having sorbed thereto a composition that canprotect against degradation of the GM stored on the solid medium. Thecomposition can also cause inactivation of microorganisms which may beassociated with a sample of GM and which may be potentially pathogenicto human handlers of the stored sample of GM.

[0037] A solid medium and a composition sorbed to a solid matrix aredisclosed in U.S. patent application Ser. No. 08/159,104 that has beenincorporated herein by reference.

[0038] As used herein, the term “storing”, “storage”, “stored” and otherderivatives of “store,” when referring to GM in dry form entrained tothe dry solid medium means the preservation of GM in a form suitable forsubsequent analysis and which has not undergone substantial degradation.The time period for which GM may be stored according to the inventionmay be as short as the time necessary to transport a sample of GM fromthe place of collection of the sample to the place where subsequentanalysis is to be performed. The condition under which the sample of GMmay be stored on the dry solid medium of the invention varies.Typically, samples are stored at temperatures from −20° C. to 40° C. Inaddition, stored samples may optionally be stored in dry or desiccatedconditions or under an inert atmosphere. Storage may be for a fewminutes up to many years. Preferably, storage occurs for about 10minutes up to 10 years.

[0039] As used herein, the term “swab,” “swab device,” and any othersimilar derivatives of “device” means an apparatus having a head portionmade of a dry solid medium that can collect, store and preserve geneticmaterial applied thereto. The preferred embodiment has a narrow rod orcylindrical-shaped support or handle in combination with the dry solidmedium that is positioned adjacent to the support. The dry solid mediumis attached to the support in a manner including, but not limited to,wrapping the dry solid medium around the support, gluing support to thedry solid medium, completely integrating the dry solid medium onto thesupport, and any other similar methods known to those of skill in theart. The head portion has the dry solid medium shaped or formed into,but not limited to, a filter, a mesh, woven bulb, and any other similarshape or form known to those of skill in the art.

[0040] In another embodiment of the invention, the dry solid medium mayfurther include a component which is functional in the subsequentanalysis to be performed on the stored GM. Subsequent analysis which maybe performed on a sample of GM stored on the dry solid medium includesanalysis methods known in the art, for example, polymerase chainreaction (PCR), ligase chain reaction (LCR), reverse transcriptaseinitiated PCR, DNA or RNA hybridization techniques including restrictionfragment length polymorphism (RFLP) and other techniques using geneticor DNA or RNA probes, genomic sequencing, enzymatic assays, affinitylabeling, method of detection using labels or antibodies and othersimilar methods. In a preferred embodiment, the dry solid medium of theinvention is a suitable medium for storage of components for subsequentanalysis, which are included on the dry solid medium. In addition, theinventors recognize that may new analytical and diagnostic methods maybe developed in the future for which the dry solid medium and method ofthe invention may be equally useful and which would fall within thespirit and scope of the claims appended hereto.

[0041] In the case of stored RNA, particularly unstable RNA, componentsfor subsequent analysis that may be included may also provide protectionagainst RNA degradation. This includes RNase inhibitors andinactivators, proteins and organic moieties that stabilize RNA orprevent its degradation.

[0042] Once the GM has been collected on the dry solid medium, it may beencased in a protective material, for example, a plastic film, which mayfurther protect against GM degradation during storage. Subsequentanalysis of the GM stored on the solid medium of the invention may beperformed in situ on the solid medium or, alternatively, the GM mayfirst be removed form the solid medium prior to subsequent analysis.

[0043] I. The Dry Solid Medium

[0044] The dry solid medium of the invention includes a compositionsorbed to a solid matrix. As used herein, the term “sorb” means that thecomposition of the invention is absorbed, adsorbed or otherwiseincorporated into or onto the solid matrix in such a way as not to bereadily removed from the matrix unless subjected to conditions which areintentionally or inadvertently performed to remove the sorbedcomposition from the solid matrix.

[0045] A solid matrix suitable for the dry solid medium and method ofthe invention includes any material to which the composition will sorband which does not inhibit storage or subsequent analysis of the GMapplied to the dry solid medium. This includes flat dry matrices or amatrix combined with a binder to form a pellet or tablet to whichcomposition is sorbed. In one preferred embodiment, the solid matrix isof a porous nature to provide entrainment of the GM onto the dry solidmedium. As used herein the term “entrain”, means that during storage theGM is bound to the dry solid medium without substantial reliance onionic, covalent or van der waals interactions. A solid matrix suitablefor this purpose includes, but is not limited to, a matrix which iscellulose based (e.g., cellulose, nitrocellulose orcarboxymethylcellulose papers), hydrophilic polymers including synthetichydrophilic polymers (e.g., polyester, polyamide, carbohydratepolymers), polytetrafluroethylene (Empore™, 3M, St. Paul, Minn.),fiberglass and porous ceramics.

[0046] GM may also be collected on a solid matrix that lacks thebelow-described composition of the invention. In addition, a componentfor subsequent analysis of a sample of GM may also included on a solidmatrix that lacks the composition of the invention. Furthermore,hemoglobin or proteins associated with a sample of GM may be removedfrom a sample of GM stored on a solid matrix which does or does notinclude a component for subsequent analysis of the stored GM using anaqueous or non-aqueous (e.g., below-described single phase phenol wash)extraction procedure. However, by using only a solid matrix for storage,the GM protecting and pathogen inactivation effects of the compositionof the invention obviously will not be available to the GM or componentsfor subsequent analysis.

[0047] To form the dry solid medium of the invention, a composition thatprotects against degradation of GM is sorbed to the solid matrix. Asused herein, the phrase “protects against degradation of GM,” means thatthe dry solid medium of the invention maintains the stored GM in asubstantially non-degraded form. This provides a sample of GM suitablefor many different types of subsequent analytical procedures. Protectionagainst degradation of GM may include protection against substantialdamaging of GM due to GM damaging events such as that caused by chemicalor biological means including action of bacteria, viruses, freeradicals, nucleases, ultraviolet radiation, oxidizing agents and acidicagents (e.g., pollutants in the atmosphere).

[0048] The composition sorbed to the solid matrix to form the dry solidmedium of the invention may include one or more of a weak base, achelating agent, or any anionic detergent or surfactant. In addition,the composition sorbed to the dry solid medium may also include uricacid or a urate salt.

[0049] As used herein, the “weak base” of the composition may be a Lewisbase that has a pH of about 6 to 10, preferably about pH 8 to 9.5. Onefunction of the weak base is to act as a buffer to maintain acomposition pH of about 6 to 10, preferably about pH 8.0 to 9.5, forexample, pH 8.6. Hence, a weak base suitable for the composition of theinvention may, in conjunction with other components of the composition,provide a composition pH of 6 to 10, preferably, about pH 8.0 to 9.5.Suitable weak bases according to the invention include organic andinorganic bases. Suitable inorganic weak bases include, for example, analkali metal carbonate, bicarbonate, phosphate or borate (e.g., sodium,lithium, or potassium carbonate). Suitable organic weak bases include,for example, tris-hydroxymethyl amino methane (Tris), ethanolamine,triethanolamine and glycine and alkaline salts of organic acids (e.g.,trisodium citrate). A preferred organic weak base is a weak monovalentorganic base, for example, Tris. The Tris may be either a free base or asalt, for example, a carbonate salt.

[0050] Although the inventors do not wish to be limited to a singletheory, it is believed that the weak base may provide a variety offunctions, including protecting the GM from degradation. In addition toproviding a buffer system, it is also believed that the weak base canact to ensure proper action of the below-described chelating agent inbinding divalent metal ions. In addition, the weak base may also preventthe action of acid nucleases that may not be completely dependent ondivalent metal ions for functioning.

[0051] The composition of the dry solid medium can also including achelating agent. According to the invention, a preferred chelating agentis a strong chelating agent is a strong chelating agent. By “strong”chelating agent it is meant that the agent binds multivalent metal ionswith a comparable or better affinity than ethylene diamine tetraaceticacid (EDTA). A preferred chelating agent according to the invention isEDTA. Although the inventors do not wish to be limited to a particulartheory, it is believed that one function of the chelating agent of theinvention is to bind divalent ions which if present with the stored GMmay partake in causing damage to the GM. Ions that may be chelated bythe chelating agent include divalent active metal ions, for example,magnesium and calcium, and transition metal ions, for example, iron.Both calcium and magnesium are known to promote GM degradation by actingas co-factors for enzymes that may destroy GM (e.g., most knownucleases). In addition, transition metal ions, such as iron, mayreadily undergo oxidation and reduction and damage nucleic acids by theproduction of free radicals or by direct oxidation.

[0052] The composition of the dry solid medium can further include ananionic detergent or surfactant. As used herein, the terms “surfactant”and “detergent” are synonymous and may be used interchangeably. Althoughnot wishing to be limited to a single theory, it is believed that theanionic surfactant of the invention functions to denature non-GMcompounds, for example, proteins, which are associated with the storedGM. Accordingly, denaturation of protein is one function of the anionicsurfactant. According to the invention, any anionic surfactant thatbinds to and denatures proteins may be suitable for the invention. Apreferred anionic detergent is a strong anionic detergent. As usedherein, a “strong” anionic detergent includes a hydrocarbon moiety,aliphatic or aromatic, containing one or more anionic groups.Particularly preferred anionic detergents suitable for the inventioninclude sodium dodecyl sulphate (SDS) and sodium lauryl sarcosinate(SLS). In a preferred embodiment, the anionic detergent of the inventioncauses inactivation of most microorganisms that have protein or lipidsin their outer membranes or capsids, for example, fungi, bacteria orviruses. This includes microorganisms that may be pathogenic to humansand are present in a sample of GM.

[0053] Inactivation of a microorganism is believed to result fromdestruction of the secondary structure of its external proteins,internal proteins and any protein containing membranes necessary forviability. The inventors recognize that the anionic detergent may notinactivate some forms of organisms, for example, highly resistantbacterial spores and extremely stable enteric virions. Moreover, theinventors further recognize that while it may be desirable to inactivatepathogenic microorganisms, the GM of a microorganism associated with thestored sample of GM is also amenable for storage on dry solid medium ofthe invention. This allows for storage and/or subsequent analysis of theGM of a microorganism associated with a stored sample of GM.

[0054] The composition invention may optionally include a uric acid orurate salt. According to the invention, the longer the period of timefor which the GM is to be stored the more likely that uric acid or aurate salt may need to be included in the composition sorbed to thesolid matrix. However, even if the GM is only to be stored for a matterof minutes, it may still be desirable to incorporate uric acid or uratesalts into the composition.

[0055] While the inventors do not wish to be limited to any singletheory, it is believed that the uric acid or urate salt may provide manyfunctions. For example, the uric acid or urate salt may be converted toallantoin in acting as a free radical trap that preferentially acceptsfree radicals that would otherwise damage the nucleotide guanine. Thefree radicals are believed to be generated by spontaneous oxidation ofthio groups that are present, for example, in denatured serum protein ofblood. Free radicals may also be generated due to oxidation or reductionof iron in blood. Because uric acid is a weak acid, it may also functionas a component of the buffering system provided by the weak base asdiscussed above. In addition, the uric acid and urate salt may act as anerodible surface in that it is sparingly soluble so that a DNA sampledried onto its crystals will be released as the urate beneath erodes.Hence, the uric acid or urate salts may also provide for easy removal ofa stored sample of GM if in situ processing is not desired.

[0056] Furthermore, after the sample of GM is applied to the dry solidmedium, the dry solid medium with the applied sample of GM may beencased in a protective material, for example, a plastic film, which mayfurther protect against degradation of stored GM. Examples of plasticfilms that are suitable according to the invention include polystyrene,polyethylene, polypropylene and other suitable lamination plastics.Encasing the dry solid medium in a protective material may beaccomplished by method known in the art. One simple method for encasingthe dry solid medium in a plastic film is to put the dry solid mediuminto a container, e.g., a polyethylene bag, which is of sufficient sizeto hold the dry solid medium such that when a plastic film in liquidform is added to the container all parts of the dry solid medium will becoated by the liquid. The plastic film, in liquid form, is added to thecontainer to coat the dry solid medium. The liquid plastic film isallowed to dry to provide a plastic film coating which encases the drysolid medium. Prior to analysis, the plastic film is removed from thedry solid medium using methods known in the art, for example, dissolvingwith organic solvents such as chloroform or mechanical stripping.

[0057] The inventors further note that a dry solid medium includingcomponents for subsequent analysis (discussed below), with an appliedsample of GM, may also be encased in a protective material as describedabove.

[0058] II. Subsequent Analysis of Genetic Material Stored on the DrySolid Medium

[0059] As used herein, “subsequent analysis” includes any analysis thatmay be performed on a sample of GM stored on the dry solid medium. TheGM stored on the dry solid medium may be analyzed in vitro. The GM mayfirst be removed from the dry solid medium prior to analysis. The sampleof GM may be subjected to chemical, biochemical or biological analysis.Examples of subsequent analysis which may be performed on samples of GMstored on the dry solid medium include polymerase chain reaction (PCR),ligase chain reaction (LCR), reverse transcriptase initiated PCR, DNA orRNA hybridization techniques including restriction fragment lengthpolymorphism (RFLP) and other techniques using genetic or DNA or RNAprobes, or genomic sequencing. In addition, diagnostic procedurespresently performed on whole blood samples, for example, the Guthrietest for phenyl-ketonuria (PKU) or enzymatic tests, for example,galactosemia may be performed on blood samples stored on the dry solidmedium of the invention after first neutralizing the effects of proteindenaturing components of the dry solid medium, for example, using a“converter solution.”

[0060] GM stored on the dry solid medium of the invention may also serveas a template for subsequent analysis of the GM using PCR methods. Thisincludes PCR using DNA or RNA as a template. When RNA is the GM to beanalyzed, the RNA may serve as a template using reverse transcriptaseinitiated PCR. The DNA sequence produced from the RNA template may thenserve as a template for further PCR amplification.

[0061] If the sample of GM is in whole blood, prior to analysis by PCR,LCR, RFLP, reverse transcriptase initiated PCR, genetic probing or othertechnique, hemoglobin or proteins associated with the GM are preferablyremoved from the sample of GM stored on the dry solid medium. Hemoglobinand/or proteins associated with the GM may be removed using methodsknown in the art, for example, aqueous or non-aqueous extractionprocedures. In addition, some components of the composition of theinvention (e.g., anionic detergent) are preferably removed, usingaqueous or non-aqueous extraction procedures, prior to subsequentanalysis.

[0062] In one preferred embodiment, hemoglobin, blood proteins or otherproteins associated with a sample of GM may be removed using non-aqueousextraction procedures, for example, the below-described single-phasephenol wash method. In addition, the single phase phenol wash may befollowed with the below-described ionic aqueous alcohol wash to provideions necessary for subsequent PCR, LCR or similar analysis of GM storedon the dry solid medium. Methods for PCR, LCR, reverse transcriptaseinitiated PCR, DNA or RNA hybridization, genomic sequencing, and othermethods of subsequent analysis are known in the art.

[0063] In another embodiment of the invention, a sample of GM associatedwith proteins or hemoglobin that is stored on the dry solid medium ofthe invention may be removed using aqueous extraction. One such aqueousextraction procedure uses aqueous SDS and mercaptoethanol. Specifically,the sample of GM stored on the dry solid medium of the invention, withor without components for subsequent analysis, is washed with analkaline solution of aqueous 6% SDS at pH 8.5 to 9.0 which contains thethiol, for example, 20 mM 2-mercaptoethanol or dithiothreitol. The GM isthen washed with isopropanol to remove substantially all remaininganionic detergent (e.g., using the aqueous alcohol wash solutiondescribed below for the single-phase phenol wash).

[0064] As will be apparent to those skilled in the art, in situsubsequent analysis of the GM stored on the dry solid medium of theinvention with or without components for subsequent analysis is ofparticular benefit in automated systems. As used herein, an “automatedsystem” includes “automatic fluid delivery systems.” According to theinvention, “automatic fluid delivery systems” include hand-held androbotic fluid delivery systems. Typically, automatic fluid deliverysystems are devices that dispense and remove fluid reagents to and fromindividual wells of multi-well reaction plates. Hand-held automaticfluid delivery systems comprise a single plunger handle with multiplefluid aspirating and dispensing ends to simultaneously aspirate anddispense fluid of a fluid reaction system from single or multiplereaction vessels simultaneously. Robotic automatic fluid deliverysystems are computer operated rater than hand-held and include suchproducts as, for example, BIOMEK 2000 (Beckman Instruments, Fullerton,Calif.), Zymark Benchmate (Zymark, Hopkinton, Mass.) and ROSYS PLATO(Rapperswil, Switzerland). Automated fluid delivery systems are alsodiscussed in U.S. Ser. No. 08/320,041 that is incorporated herein byreference.

[0065] A. Single Phase Phenol Wash

[0066] Another aspect of the present invention provides a non-aqueousextraction method for removing hemoglobin and/or proteins associatedwith a stored sample of GM. According to the invention, hemoglobin andproteins may be removed form a stored sample of GM using a single-phasephenol wash. Preferably, the single-phase phenol wash also removescomponents of the composition of the invention (e.g., anionicdetergent). The single-phase phenol wash is performed by contacting asingle-phase phenol solution with the dry solid medium having appliedthereto a sample of GM. The single-phase phenol wash is removed for thedry solid medium containing a sample of GM and the dry solid medium isthen contacted with an aqueous alcohol wash. The aqueous alcohol wash isthen removed. If the sample of GM is to be analyzed by PCR, LCR or othermethod that relies on ions, the sample of GM may then be contacted withan ionic aqueous alcohol wash prior to PCR analysis. As previouslynoted, a non-aqueous wash system such as the single phase phenol wash oran aqueous wash system may also be used to wash a sample of GM stored ona solid matrix, with or without included components for subsequentanalysis, which does not have the composition of the invention sorbedthereto.

[0067] The single-phase phenol wash provides for removing proteinsand/or hemoglobin or components of the composition of the dry solidmedium that may affect the subsequent treatment to be performed on theGM stored on the dry solid medium without affecting the stored GM.Unlike prior art methods, the present wash system does not require twophase separations (aqueous and organic phase) during processing andtends not to dissolve GM present in blood. The single-phase wash is alsoamenable to automated systems.

[0068] The single-phase phenol wash is also disclosed in U.S. patentapplication Ser. No. 08/159,104. The wash includes two solutions,referred to below as “single phase phenol solution” (solution A) and“aqueous alcohol wash solution” (solution B). A third solution, “ionicaqueous alcohol wash solution” (solution C), may be used, after thesingle phase phenol wash if the sample of GM is to be analyzed using amethod which requires additional ions for example, PCR and LCR.

[0069] Solution A:

[0070] The single-phase phenol solution (also referred to as waterunsaturated phenol wash) includes phenol and hydroxyquinoline saturatedwith Tris-acetate and mercaptoethanol. Preferably, 50 grams (gm) ofphenol is combined with 120 milligram (mg) of 8-hydroxyquinolonesaturated with 10 milliliter (ml) of 1.0 molar (M) Tris-acetate at pH6.0 to 9.0, preferably pH 8.0, and 1.0 ml 2-mercaptoethanol. Aftersaturation by shaking at a temperature of about 0° C. to 20° C.,preferably 0° C. to 4° C. the aqueous phase is removed and discarded.

[0071] Solution B:

[0072] The aqueous wash solution utilizes any C₂-C₅ alcohol, such asisopropanol or n-propanol, or any other similar water miscible solventcombined with an acetate salt or similar organic soluble salt whichprovides a pH of about 7.0 to 9.0, preferably about pH 7.8. In apreferred embodiment 75% by volume isopropanol is combined with 25% byvolume of 0.1 M potassium acetate at a pH of about 7.8.

[0073] Solution C:

[0074] The ionic aqueous wash solution is a wash solution which providesappropriate ions, for example, magnesium, manganese, sodium, protamine,tris and similar ions which may be useful in a subsequent analysisprocedure. For PCR analysis, magnesium is preferred. Accordingly, theionic alcohol wash solution utilizes any C₂-C₅ alcohol, such asisopropanol and n-propanol, or any other similar water miscible solventcombined with an acetate salt or similar organic soluble salt whichprovides a pH of about 6.0 to 8.5, preferably about pH 7.8. In apreferred embodiment for magnesium ions, 75% by volume isopropanol iscombined with 25% by volume of 0.01M magnesium acetate at a pH of about7.5.

[0075] According to the invention, a sample of GM associated withhemoglobin and/or protein which is stored on the dry solid medium iscontacted with solution A for about 15 minutes to about 3 house,preferably about 1.5 to about 2 house, at a temperature which is greaterthan 37° C., preferably about 45-65° C., more preferably about 50° C.The temperature at which the sample of GM is contacted with solution Ais not critical if the dry solid medium of the invention does not alsoinclude a component for subsequent analysis, such as a primer, which isdiscussed below. The solution A is then removed. Preferably, the sampleof GM is then quickly washed 1-4 times, for a few seconds each time withfresh solution A.

[0076] After removal of solution A, the sample of GM is washed, for afew seconds, 1-4 times with solution B. Preferably, the solution Bwashes are at about 10° C. to 25° C. If solution C is to be used, aftersolution B is removed, solution C is applied to the sample of GM forabout 1-60 minutes, preferably about 30 minutes, at a temperature ofabout 10° C. to 25° C. The solution C is removed and the sample of GM onthe dry solid medium is dried.

[0077] Generally, the single-phase phenol wash of the invention providesa substantially protein and/or hemoglobin free GM that provides a veryconvenient starting point for a variety of analytical procedures basedon nucleic acids. The use of the single-phase phenol wash and ionicaqueous alcohol wash is further described in the Examples.

[0078] According to the invention, the single-phase phenol wash may beused to provide a substantially protein/hemoglobin free sample of GMstored on any solid system. Moreover, as described below, thesingle-phase phenol wash is of particular benefit when a solid storagesystem incorporates components for subsequent analysis such as primers.Hence, the single phase phenol wash is useful to remove proteins and/orhemoglobin from a sample of GM stored on a storage medium regardless ofwhether or note the storage medium incorporates the GM protectingcomposition of the invention and regardless of whether or not thestorage medium includes components necessary for subsequent analysis.

[0079] B. Dry Solid Medium Including Components for Subsequent Analysisof Genetic Material

[0080] In another embodiment of the invention, the dry solid mediumincludes components for subsequent analysis of a stored sample of GM. Aspreviously stated “subsequent analysis” means any analysis of which maybe performed on a sample of GM stored on the dry solid medium. Thisincludes chemical, biochemical or biological analysis. Examples ofsubsequent analysis which may be performed on samples of GM stored onthe dry solid medium include PCR, LCR, reverse transcriptase initiatedPCR, DNA or RNA hybridization techniques including RFLP and othertechniques using genetic or DNA or RNA probes, genomic sequencing,enzymic assays, affinity labeling, method of detection using labels orantibodies and other methods known in the art.

[0081] As used herein, “components for subsequent analysis of a storedsample of GM” includes a molecule, compound, reagent, or other componentthat may be functional in analyzing, or preserving for analysis, asample of GM, or analysis of some other component stored with the GM onthe dry solid medium. In some instances, subsequent analysis of someother component stored with the GM on the dry solid medium. In someinstances, subsequent analysis will involve an analytical method that isused to establish the absence or presence of a particular geneticsequence. The genetic sequence to be analyzed is termed a “targetsequence.” The target sequence or other region of a sample of geneticmaterial may serve as a “template” for PCR amplification.

[0082] One or more component may be included on the dry solid medium.The component(s) included on the dry solid medium is dependent on theparticular analysis to be performed on the sample of GM. The advantageof including a component functional in the subsequent analysis of asample of GM is readily apparent in situations where large numbers ofsamples of GM are simultaneously analyzed using automated systems. Anexample of a component that may be included on a dry solid medium is a“nucleotide sequence.”

[0083] As used herein, nucleotide sequence includes a primer, (e.g., aPCR primer, a primer for reverse transcriptase initiated PCR), sets ofoligonucleotide substrates for LCR, DNA or RNA probe, oligonucleotidesused in genetic sequencing, or target sequence stabilizer.

[0084] As used herein, a “primer” means one pair of oligonucleotides,each of which is capable of annealing to a complementary nucleic acidsequence in a sample of GM, and which are oriented so that each primerwill prime the synthesis of a strand of DNA (or conceivably, a strand ofRNA) containing a sequence complimentary to the other oligonucleotide.For some applications, it is desirable that the sequence of theoligonucleotide and its complimentary sequence are predetermined. Inother applications, the sequence may be unknown. A plurality ofdegenerate primers or primers with sequences predicted to bind to thecomplimentary sequence may be used. The primer-pair may be used toamplify a pair of complimentary sequences and the sequence between thetwo complimentary sequences of known or unknown size. In addition, theprimer may be used to amplify a template that is initiallydouble-stranded DNA or RNA, as well as single-stranded DNA or RNA. For asingle-stranded template, it is understood that the sequencecomplimentary to one of the oligonucleotides will be synthesized byelongation of the first primer as it is annealed to its complementarysequence.

[0085] Primers for PCR or for reverse transcriptase initiated PCR may bepresent as a primer-pair, as is usual, or there may be only one primeras may be the case with genomic-sequencing or some protocols for reversetranscriptase initiated PCR.

[0086] In another embodiment of the invention, a component forsubsequent analysis may be a nucleotide sequence referred to as a“target sequence stabilizer.” A target sequence stabilizer may bebeneficial for enhancing storage of an unstable single strand RNA or DNAstored as a sample of GM on the dry solid medium. According to thisembodiment, a “target sequence stabilizer” is a nucleotide sequencewhich may be complimentary to an entire region of RNA or DNA which is tobe analyzed. As is known in the art, many single strand RNAs areunstable and vulnerable to degradation by low levels of omnipresentRNases. However, the inventors believe that by including a targetsequence stabilizer on the dry solid medium, the target sequencestabilizer binds to the single strand RNA (or DNA) sample and the“hybrid” formed is less vulnerable to degradation. For example, it isknown in the art that double-stranded RNA/DNA hybrids are highlyresistant to the common RNases. The RNA or DNA annealed to the targetsequence stabilizer may then be further analyzed using methods known inthe art, for example, LCR or combinations of LCR and reversetranscriptase initiated PCR.

[0087] Therefore, according to the invention, components for subsequentanalysis of a stored sample of GM includes components which function insubsequent analysis of a sample of GM as well as provide furtherprotection of a stored sample of GM such that the GM is available, innon-degraded form, for subsequent analysis. The inclusion of anucleotide sequence on the dry solid medium provide significantadvantages for simplifying subsequent analysis of GM by, for example,automated PCR systems. In addition, selection of appropriate nucleotidesequences may provide additional protection from degradation of GMmaterial such as unstable RNA.

[0088] Other components for subsequent analysis of stored GM that may beincluded in the dry solid medium of the invention are, for example,genetic probes or oligonucleotide or polynucleotide sequences withlabels of diverse types. Labels may include a fluorescent chemicalgroup, radioactive isotope, chemical binding agent (e.g., biotin),antigens or enzymes.

[0089] PCR amplification of a stored sample of GM, for example, bloodDNA, is greatly simplified using the dry solid medium of the inventionincluding a nucleotide sequence such as a primer. Furthermore, utilizingthe single phase phenol wash of the invention to remove proteins and/orhemoglobin associated with a sample of GM on a dry solid medium whichincludes a nucleotide sequence, provides for insubstantial loss of theincluded nucleotide sequence. By “insubstantial” it is meant that thewash, when completed, has not caused sufficient loss of primer toprevent PCR, LCR, reverse transcriptase initiated PCR, probing, etc.Hence, because both the nucleotide sequence and sample of GM remainentrained to the dry solid medium during the washing procedure, in situprocessing is possible. This provides a significant advantage for GManalysis using automated systems.

[0090] In the case of PCR amplification, for example, by including aprimer necessary for amplifying a specific DNA or RNA gene sequence,multiple samples containing different primers may be analyzed using asingle PCR reaction mixture. Typically, only one mixture of PCR enzymeand nucleotides to all vials is necessary. Moreover, even if differentprimers included with different GM samples require variation in reactionconditions such as magnesium ions and/or pH, these variations can beaccommodated with only two additional pump or pipette systems to deliverinexpensive salts and buffers that will cover most contingencies.

[0091] The components for subsequent analysis are preferably included onthe dry solid medium before subsequent treatment of the GM. Variouspermutations for inclusion of the components are possible. For example,the components may be added and mixed with the composition of the drysolid medium prior to application of the composition to the solidmatrix. Also, the primers may be added to the solid matrixsimultaneously with the composition. Alternatively, the composition ofthe invention may be applied to the solid matrix and the primers appliedsubsequently. In another alternative, a sample of GM may be applied tothe dry solid medium and a component applied to the dry solid mediumafter collection of the sample of GM but before removing any hemoglobinor protein associated with the GM sample. It is also foreseeable that acomponent may be added to the dry solid medium after removing proteinand/or hemoglobin but before subsequent analysis.

[0092] Thus, the invention provides for novel and previously impracticalstrategies to be made feasible by a dry solid medium including acomponent for subsequent analysis in combination with methods that allowfor co-processing the component with the stored GM.

[0093] C. Inclusion of Multiple Nucleotide Sequences on a Single DrySolid Medium

[0094] The dry solid medium of the invention may also be identified orreferred to as a card. A card may be prepared in such a manner as toprovide different components for subsequent analysis at differentlocations on a single card. When the component for subsequent analysisis a nucleotide sequence, multiple different nucleotide sequences forpriming amplification or stabilizing different target sequences may bestored on one card. As used herein, “multiple” means greater than one.For example, a single sample of GM to be analyzed may be stored on asingle card, which is about 1 mm to 5 mm square. Therefore, a card,which is about 5 cm×10 cm, could be divided, for example, into 200individual samples. Each individual 5 mm square could hold a differentnucleotide sequence. Accordingly, a single 5 cm×10 cm card could providefor subsequent analysis using 200 different nucleotide sequences.

[0095] In a preferred embodiment, a nucleotide sequence included on acard for subsequent analysis is a primer. According to this embodiment,a dry solid medium card may incorporate all or many of the primers for aparticular application on a single card. For example, one applicationmay be to screen for various target sequences, in a single GM sample,which are all involved in a particular genetic disease. Hence, anunlimited range of pre-made and pre-labeled sample-receiving cards maybe held at a collection center ready for samples of GM such as blood.These cards, including primers, may be labeled, packaged and sold forparticular categories of usage and put together indefined-purpose-folios (e.g., a card for standard penitentiary purposesor a card screening for various diseases of genetic origin). Moreover,the inclusion of a primer on the card allows the dry solid medium to beemployed in a variety of different fields and still be convenientlyprocessed together at one centralized, unspecialized center forautomated analysis. This will allow the automated system to use onebasal mixture of enzyme, and possibly buffer, to simultaneously amplifyall target sequences for which primers are present on one type of card.As previously noted, the potential variations in ions and pH can behandled through a variety of ways, for example, separate delivery pumpsor the use of the newer pellet-delivery of magnesium or other substancesin wax.

[0096] III. Swab Device:

[0097] The swab device of the present invention utilizes the dry solidmedium having the properties disclosed herein. As described in moredetail in Example 6, the swab device and the preferred embodimentdisclosed therein is used to obtain, store, and analyze various geneticmaterial samples obtained from biological and non-biological fluidsamples. This embodiment includes a head portion having a dry solidmatrix with the properties of sorbing, storing and preserving thegenetic material placed on the dry solid matrix therein. There arenumerous types of materials that compose the dry solid medium. Thepreferred embodiment utilizes polyester. Other suitable materials of thedry solid medium include, but are not limited to, cellulose basedmaterial, hydrophilic polymers, synthetic hydrophilic polymers,polyamide, carbohydrate polymers, polytetrafluroethylene, fiberglass,glass, porous ceramics and any other similar materials known to those ofskill in the art.

[0098] The swab device additionally has a solid support in combinationwith the dry solid medium. The solid support allows for the safehandling of the dry solid matrix to prevent any kind of contamination ofthe dry solid medium. The support can be made of any material including,but not limited to, plastic, wood, metal, fiberglass and any othersimilar solid, materials known to those of skill in the art.Additionally, the support can be permanently or removeably attached tothe dry solid medium through any mechanism known to those of skill inthe art. Attachment mechanisms include, but are not limited to, glue,thread, stitching, wrapping the dry solid medium around the support,encasing the dry solid medium, and through any other similar methodsknown to those of skill in the art.

[0099] The swab device can be used in any number of clinical andnon-clinical settings. Additionally, the swab device can obtain samplesfrom numerous organic or inorganic, and living or nonliving surfaces.The swab device is safe to be used inside living organisms to swabsamples from the inside of flesh wounds, inside of body orifices, andfrom any part of the human body.

[0100] There is a wide range of liquids and biological samples that thepresent invention can be used to collect. These liquids and samplesinclude, but are not limited to, buccal fluids, cerebrospinal fluid,feces, plasma, blood, lymphatic fluids, urine, suspension of cells orviruses, serum samples, and any other similar biological fluid samplesknown to those of skill in the art.

EXAMPLES Example 1 In situ PCR Amplification of Blood DNA Stored on DrySolid Medium

[0101] A sample of blood DNA stored on the dry solid medium of theinvention was amplified in situ by the polymerase chain reaction (PCR)technique. The dry solid medium used in this Example was cellulose basedpaper having sorbed thereto a composition comprising, per sq. cm ofpaper, 2 micromols uric acid, 8 micromols tris (free base), 0.5micromols EDTA and 1 mg SDS. The stored blood was washed and suitableions added prior to PCR amplification.

[0102] 1.1 Single Phase Phenol Wash

[0103] A. Materials

[0104] Solution A: Single-phase phenol wash solution. A suitable mixtureis phenol, 50 gm containing 120 mg of 8-hydroxyquinoline that has beensaturated with 10 ml of 1.0 M tris-acetate pH 8.0 and 1.0 ml2-mercaptoethanol. After saturation by shaking at room temperature, theaqueous phase is thoroughly removed and discarded.

[0105] Solution B: Aqueous alcohol wash solution. 75% v/v Isopropanol,25% v/v 0.1M potassium acetate at pH 7.8.

[0106] Solution C: Ionic aqueous wash solution. 75% v/v Isopropanol, 25%v/v 0.01 M magnesium acetate.

[0107] B. Method

[0108] All steps are preferably carried out in a single tube made of asuitable phenol resistant material, e.g. polyethylene.

[0109] 1. Removal of protein: a 5 mm×5 mm square of blood impregnateddry solid medium was treated with 1 ml of solution A, for approximately1.5 hours at 45° C. (since no primers were present, this temperature andtime is not critical). The solution A was then aspirated to waste andthe paper square quickly washed three times with 0.25 ml of solution A.Each wash was for only a few seconds and was immediately aspirated towaste.

[0110] 2. Removal of phenol and addition of suitable ions: the dry solidmedium from step (a) above was rapidly washed three times with 1 ml ofsolution B. Washes were at room temperature and were simple additionsfollowed by aspiration to waste. The paper was then washed for 20minutes at room temperature with solution C. (This is to saturate the GMon the paper with magnesium ions and remove the last of the phenol). Thesolution C is aspirated to waste and the dry solid medium wassolvent-dried with one wash of pure isopropanol and then vacuum dried.

[0111] The washed dry solid medium was relatively quite white withoutany obvious remnants of the red-brown color of blood. It was then usedin a PCR reaction mix.

[0112] 1.2 Amplification of DNA on Dry Solid Medium

[0113] The washed sample on dry solid medium described above has beenshown to be a suitable substrate for DNA polymerase chain reaction (PCR)amplification of DNA.

[0114] A. Materials

[0115] 1. Specimens

[0116] Extracted DNA: 10 ml of blood was removed from a male volunteerusing standard protocols.

[0117] Dry solid medium: Blood samples from the same volunteer wereapplied directly to the dry solid medium with subsequent single phasephenol washing as described above. The dry solid medium was cut intoabout 1 mm×1 mm pieces for use in PCR reactions.

[0118] 2. Targets for Amplification

[0119] Target No. 1: Region of exon 2 of the n-Ras proto-oncogene onchromosome 1. The primers used are: R1: 5′ TGA CTG AGT ACA AAC TGG TGGTG 3′ (SEQ ID NO:1) R2: 5′ CTC TAT GGT GGG ATC ATA TTC A 3′ (SEQ IDNO:2).

[0120] The amplified DNA fragment obtained with these primers is 110 bpin size.

[0121] Target No. 2: A male specific Y chromosome repeat sequence.

[0122] The primers are: 007: 5′ TGG GCT GGA ATG GAA AGG AAT GCA AAC 3′(SEQ ID NO:3) and 008: 5′ TCC ATT CGA TTC CAT TTT TTT CGA GAA 3′ (SEQ IDNO:4).

[0123] The amplified DNA fragment obtained with these primers is 124 bpin size.

[0124] Target No. 3: A male specific Y chromosome repeat sequence.

[0125] The primers used are: 004: 5′ GAA TGT ATT AGA ATG TAA TGA ACT TTA3′ (SEQ ID NO:5) and 006: 5′ TTC CAT TCC ATT CCA TTC CTT TCC TTT 3′ (SEQID NO:6).

[0126] The amplified DNA fragment obtained with these primers is 250 bpin size.

[0127] B. Method

[0128] 1. PCR Protocol

[0129] Extracted DNA (1 μg) or about 1 mm×1 mm fragments of washed drysolid medium containing a sample of blood DNA were placed into 0.5 mlEppendorf tubes and made to 25 μk in PCR reaction mixture consisting of:

[0130] 67 mM Tris HC1 (pH 8.8 @25° C.)

[0131] 16.6 mM (NH₄)₂ SO₄

[0132] 2.0 mM MgC1₂

[0133] 0.01% (w/v) gelatin

[0134] 0.1 mM deoxynucleotides (dATP, dTTP, dCTP, dGTP)

[0135] 0.25 μg of each primer (for respective target)

[0136] 0.25 U of Taq DNA polymerase.

[0137] 2. The Amplification Conditions

[0138] The mixture was overlaid with 25 μl of light mineral oil and DNAamplification was performed by 30 cycles of amplification on a PerkinElmer-Cetus “thermal cycler”. The first cycle consisted of: DNAdenaturation 6 min. @ 94° C. Primer - DNA annealing 1 min. @ 55° C. TaqDNA polymerase extension 1 min. @ 75° C.

[0139] followed by 29 cycles as above except DNA denaturation was for 1minute @ 94° C. and the extension time on the last cycle was 10 minutes@ 72° C. before cooling the reaction mixture to 4° C.

[0140] 1.3 Results

[0141] After amplification, 10 μl aliquots of PCR mixture were analyzedby electrophoresis on 15% (w/v) polyacrylamide gels. Amplified targetDNA was visualized by UV illumination of the ethidium bromide stainedgel.

[0142] An analysis of the products from PCR amplification of extractedblood DNA and blood DNA stored on dry solid medium are shown in FIG. 1,as follows:

[0143] Lanes 1-3: Target No. 1, lane 1: 1 μg DNA, lane 2: 1 mm² filter,lane 3: control (no DNA);

[0144] Lanes 4-5: Target No. 2, lane 4: 1 μg DNA, lane 5: 1 mm² filter;Lane 6: control (no DNA);

[0145] Lanes 7-9: Target No. 3, lane 7: 1 μg DNA; lane 8: 1 mm² filter,lane 9: control (no DNA);

[0146] Lane S: DNA size markers (pUC19/Hpall digest).

[0147] The results shown in FIG. 1 clearly demonstrate that the DNA hasnot been changed in any way as a result of its storage on the dry solidmedium of the invention, and that it can be used in situ as describedherein.

Example 2 PCR Reactions on DNA Stored on Dry Solid Medium IncludingPrimers

[0148] PCR analysis was performed on dry solid medium including primersadded to the dry solid medium prior to the collection of a sample ofblood DNA.

[0149] 2.1 Genetic Material

[0150] A. Materials

[0151] 1. DNA Templates

[0152] For preparing samples of genetic material containing DNA, 3 μl ofhuman blood was applied to a 3 mm square sample of dry solid medium, andthe blood was allowed to dry. Unless otherwise indicated, this 3 mmsquare was then used in each PCR reaction. 3 μl of blood corresponds toapproximately 250 nanograms of white-cell DNA. In addition, a controlsample of purified human DNA in free solution was amplified using PCR.

[0153] 2. Primers

[0154] Two synthetic oligonucleotide primers derived from the humanClass II HLA gene, DQ-alpha, were used in the experiments. Theirsequences are: 5′ GTGCTGCAGGTGAAACTTGTACCAG 3′ (SEQ ID NO:7)5′ CACGGATCCGGTAGCAGCGGTAGAGTTC 3′ (SEQ ID NO:8)

[0155] The size of the PCR product expected using the two primers is 262bp. Each primer was stored as a stock solution at a concentration of 50ng/μl. When added to the dry solid medium prior to processing, 150 ng ofboth primers was added to the 3 mm square of dry solid medium. Theprimers were always used together at equal concentrations.

[0156] 2.2 Single Phase Phenol Wash

[0157] Solution A: Single-phase phenol solution. 50 g of phenol,containing 120 mg of 8-hydroxyquinoline, was saturated with 10 ml of 1.0M Tris-acetate pH 8.0 and 1.0 ml of 2-β-mercaptoethanol. Aftersaturation at between 0° C. and 4° C., the aqueous phase was thoroughlyremoved and discarded.

[0158] Solution B: Aqueous alcohol wash solution. 75% v/v isopropanol,25% v/v 0.1M potassium acetate, pH 7.8.

[0159] Solution C: Ionic aqueous wash solution. 75% v/v isopropanol, 25%v/v 0.01 M magnesium acetate.

[0160] B. Method

[0161] 1. Washing the Dry Solid Medium

[0162] Samples of genetic material stored on the dry solid mediumincluding a primer were processed by first washing the medium with 200μl of Solution A for two hours at either 37° C. or 50° C., followed bytwo quick rinses of the aqueous phase with Solution A. The medium wasnext rinsed three times for five minutes each with 200 μl of Solution Bat room temperature. The medium was then washed with 200 μl of SolutionC for 20 minutes at room temperature and then dried under vacuum. (Thewash times were all in excess. In practice, shorter times may be used.The effectiveness of the washes may be monitored by observing loss ofcolor: (1) the hemoglobin; and (2) the hydroxyquinoline.)

[0163] 2.3 PCR Protocol

[0164] A. Materials

[0165] 1. The Example PCR Mix 50 μl.

[0166] The following mixture was placed on washed dry solid mediumcontaining a sample of blood before heat-cycling:

[0167] 67 mM tris-HC1, (pH 8.8 @25° C.)

[0168] 16.6 mM (NH₄)₂ SO₄ 0.2 mg/ml gelatin

[0169] 0.2 mg/ml gelatin

[0170] 0.45% Triton X-100

[0171] 2.0 mM MgCl₂

[0172] 0.2 mM each of dATP, dTTP, dCTP, dGTP

[0173] 2.5 units Taq polymerase.

[0174] Template blood DNA was on dry solid medium or purified human DNAas free solution. (50 ng template added when in free solution.)

[0175] As shown in table 1, primers were included with the dry solidmedium and/or in free solution. (100 ng each primer if added as freesolution.)

[0176] B. Methods

[0177] 1. The Amplification Conditions

[0178] 4 minutes @ 94° C. (start conditions)

[0179] 1 minute @ 94° C.

[0180] 40 seconds @ 50° C. (32 cycles)

[0181] 1 minute @ 72° C.

[0182] 4° C. (soak-end conditions)

[0183] The expected product is a 242 bp segment of the human class-IIHLA gene, DQ-alpha.

[0184] 2. Electrophoresis Conditions

[0185] Electrophoresis was in 1% agarose, 0.045 M tris, 0.045 M Boricacid, 1 mM EDTA, pH 8.0, 1.5 hours at 8.5 volts/cm.

[0186] C. Results

[0187] A 3 mm×3 mm primer-loaded dry solid medium were taken. 3 μl offreshly collected human blood from one donor was placed on all dry solidmedium samples and allowed to dry. It was then stored at roomtemperature for 7 days.

[0188] The squares of dry solid medium were then washed using thesingle-phase phenol wash and all media-squares then dropped into PCRmixes either with or without added primers. There were thus trials inwhich the primers were added to the medium before loading blood to themedium, after loading blood to the medium, and to the final PCR mix (seeTable 1).

[0189] After the cycling 8 μl samples of each reaction mix wereelectrophoresed with Promega PCR marker mix (Promega Corp., Madison,Wis.). (Bands at 1000 bp, 750 bp, 500 bp, 300, 150, 50). The gel wasstained with ethidium bromide and the product bands photographed (seeFIG. 2).

[0190] Four samples were processed with the single-phase phenol washtaking place at 37° C., and four samples were processed with the singlephase phenol wash taking place at 50° C. Subsequent processing steps andPCR reaction conditions for the two sets of samples were identical. FIG.2 shows that extractions performed at 37° C. generated bands of thepredicted size, and whose intensities varied (lanes 1-4), whileextractions performed at 50° C. yielded intense bands of the predictedsize (lanes 5-8). To show that the PCR products on DNA recovered fromthe dry solid medium were specific and corresponded to the expected PCRproduct, PCR reactions were performed on human DNA purified usingstandard methods (FIG. 2, lane 12), or using no DNA (FIG. 2, lane 13).No product was observed if no exogenous DNA was added to the reaction(FIG. 2, lane 13), and the product obtained with purified DNA comigratedwith the PCR product obtained from processed samples of medium (FIG. 2,compare lanes 11 and 12).

[0191] Two additional sets of samples were then extracted at 37° C. Toone reaction primers were added to the dry solid medium after the blood.To a second reaction primers were likewise added to the dry solid mediumafter the blood, and additional primers were added just prior to thebeginning of the PCR reaction. The results of this experiment are shownin FIG. 2, lanes 9 and 10, respectively. Lane 9 reveals that the DNA onthe dry solid medium generated a PCR product of the expected size whenprimers are added to the dry solid medium after the collection of theblood sample. A more intense PCR product of the expected size wasobtained when primers were added just prior to performing the PCRreaction (compare lanes 9 and 10). The results of these experiments aresummarized in Table 1. TABLE 1 Experimental Conditions of PCR Reactionson DNA Stored on Dry Solid Medium Source of Time of Template Addition ofPhenol Wash Lanes in Mean Results DNA Primers Temperature of PCR Humanblood A 37° C. 1-4 ++ on washed dry solid medium Human blood A 50° C.5-8 ++++ on washed dry solid medium Human blood A 50° C. 11 ++++ onwashed dry solid medium Purified D N/A 12 ++++ human DNA in solution NoDNA D N/A 13 — Human blood B 37° C.  9 +++ on washed dry solid mediumHuman blood C 37° C. 10 ++++ on washed dry solid medium

[0192] In summary, the use of the dry solid medium containing primers isclearly practical, it opens a window of technique that makes large-scalePCR analysis much more practical and amenable to less skilled labor, andopens potential new applications, with unstable templates.

Example 3 In situ Digestion of Stored Blood with RestrictionEndonuclease Eco R1

[0193] To determine if samples of blood DNA stored on a cellulose-baseddry solid medium were digestible in situ with restriction endonucleases,and thus suitable for procedures such as RFLP analysis, samples of bloodwere collected on cellulose-based dry solid medium, washed with singlephase phenol wash and digested with the restriction enzyme Eco R1. Inthis experiment, samples of blood were stored on the dry solid mediumfor as long as 18 months prior to processing.

[0194] 3.1 Single Phase Phenol Wash

[0195] A. Materials and Methods

[0196] The single-phase phenol wash and other wash solutions aredescribed in Example 1 and 2. 1.5 ml of solution A was added to a dried10 mm×10 mm piece of dry solid medium containing approximately 0.1 ml ofdried human blood, and the solution was mixed using a standardlaboratory mixer and incubated at 37° C. for 30 minutes. The solution Awas then removed by aspiration and fresh solution A added. Treatmentswith solution A were repeated until all the heme color disappeared fromthe dry solid medium.

[0197] Next, the samples were washed five times with solution B for twominutes each, then twice with solution C, also for about two minuteseach. Between each wash, the medium sample was subjected to centrifugalstripping. For this the medium was briefly spun in a small microfugetube in which a hole had been pierced and which had been nested inside alarger microfuge tube. The medium was then dried under vacuum or,alternatively, by centrifugal stripping. Following the processing stepsthe samples that initially were black after application of the blood andsubsequent drying, were white.

[0198] 3.2 Digestion of Samples Stored on Dry Solid Medium UsingRestriction Enzymes

[0199] A. Material

[0200] Restriction enzyme Eco R1 was used in buffers supplied byBoehringer Manheim.

[0201] B. Method

[0202] The washed dry solid medium was moistened with 50 μl ofrestriction enzyme buffer containing an excess of restriction enzyme.For digestions with the restriction enzyme Eco R1 , 60 units of enzymewere added. To ensure complete wetting, the medium was wet in thebuffer, centrifugally stripped, and re-moistened with the buffer andenzyme mix. Digestions proceeded for 2.5 hours at 37° C.

[0203] The restricted DNA was stripped from the medium by firstcentrifugally stripping the medium and collecting the stripped fluid.DNA was then further removed by washing the solid medium with 25 μl of7.5M ammonium acetate, followed by a wash with 25 μl of 2.5M ammoniumacetate. At each wash step the medium was suggested to centrifugalstripping in order to maximize recovery of the fluids. The fluids fromthe wash steps were combined in a final volume of about 100 μl. The DNAwas precipitated from the collected fluid by adding 250 μl of ethanol,and air-drying the final pellet. The pellet was resuspended in 20 μl ofwater, and 10 μl of the resuspended pellet was used in gelelectrophoresis.

[0204] 3.3 Gel Electrophoresis

[0205] A. Conditions for Gel Electrophoresis

[0206] Digestion products were electrophoresed in a 1.5% agarose gelprepared in Tris-Acetate (composition was 0.01M Tris, 0.005M sodiumacetate, 0.5M EDTA adjusted to pH 7.8 with acetic acid) at 50V, 80 mAfor two hours, after which the gel was stained with ethidium bromide andphotographed. (See FIG. 3).

[0207] 3.4 Results

[0208] To determine if blood samples stored on media of the inventionfor extended periods of time could be digested with restriction enzymes,samples of cellulose-based dry solid medium to which blood had beenadded 19 months previously, 11 months previously, or two weekspreviously were processed and then digested with Eco R1. FIG. 3 containsa photograph of the ethidium staining pattern and reveals a smearingpattern characteristic of an Eco R1 digest of human DNA. Thus, DNAstored on the medium for at least 19 months is digestible withrestriction endonucleases.

Example 4 Inactivation of Herpes Simplex Virus Type I and otherMicroorganisms, by the Dry Solid Medium

[0209] In these experiments dry solid medium prepared according to theinvention was tested for its ability to inactivate herpes simplex virus,type I (HSV). HSV-type I was chosen because it is relatively resistantto inactivation and is transmissible through blood and other bodilyfluids.

[0210] 4.1 Isolation of HSV Virus

[0211] A. Materials and Method

[0212] A recent clinical isolate of HSV type I recovered from an eyelesion was grown to high titer (>10⁸ plaque forming units/ml) in tissueculture flasks of Hep2 epithelial and MRC-5 fibroblast cells, harvestedoff the flasks, and sonicated to release intracellular virus. Viralparticles were concentrated by high-speed centrifugation of a clarifiedsupernatant of the sonicate and were resuspended in viral transportmedium (VTM) containing 0.2M sucrose in a 0.02M phosphate buffer (pH7.6), 10% fetal calf serum, and antibiotics.

[0213] 4.2 Application of Viral Particles to the Dry Solid Medium andSubsequent Recovery

[0214] For experiment I of Example 4, the crude viral preparation wasdiluted 1:10 in either VTM or in fresh, heparinized blood. In 10 μlsample of the dilution was tested in duplicate on both untreated 10mm×10 mm pieces of untreated cellulose based paper and also on 10 mm×10mm square pieces of dry solid medium prepared according to theinvention. After application of virus, the squares were supported in airby fine forceps, and after the appropriate time interval, the virus waseluted from the squares by vigorous agitation in 1 ml of MRC-5maintenance medium (MM) (Eagles MEM based medium with Earle's salts)containing 2% fetal calf serum.

[0215] In experiment II of Example 4, a stock virus was taken fromstorage at −70° C. and diluted 1:4 in citrated blood.

[0216] 4.3 Assaying Viral Infectivity

[0217] A. Material and Method

[0218] The elute was tested for infectivity using a fluorescent focusassay. Samples were diluted 1:100 in MM, and 0.2 ml of the dilution wasthen added to a well of MRC-5 fibroblasts in a 48-well cluster tray.After a 45 minute incubation at 37° C., the sample was replaced with 0.5ml and the tray was incubated overnight at 37° C. in the presence of 5%CO2. Cells were then fixed, stained with fluorescein-conjugated antibodyto HSV, and the number of fluorescent foci was counted under areverse-stage fluorescent microscope.

[0219] 4.4 Results and Discussion

[0220] HSV type 1 viral particles were added either to untreatedcellulose based paper or to cellulose based paper treated according tothe invention to produce the dry solid medium, and then eluted aftervarying lengths of time. In one experiment, the virus was stored in VTM,and in the second the virus was resuspended in blood. The results of theexperiment are shown in Table 2. In both experiments, viral infectivitywas reduced to undetectable levels—in these conditions, greater than99.5% inhibition—when virus was added to the dry solid medium, even whenthe viral particles were eluted immediately. Viruses in blood elutedfrom untreated cellulose based paper (CBP), in contrast, were onlyslightly, if at all, inhibited. A similar inhibitory effect on HSVinfectivity was observed when the experiments were performed withviruses recovered from a frozen stock.

[0221] The invention has also shown the inhibitory effect of the drysolid medium on growth of Staphlococcus aureus, Staphlococcusepidermidis and adventitious microorganisms in the environment. TABLE 2Infectivity of HSV Following Application on to Untreated cellulose basedpaper (CBP) or on to Dry Solid Medium. Viral Titer B. HSV Viral TiterPercent in Percent Treatment A. HSV in VTM Inhibition Blood InhibitionUntreated 1.9 × 10⁷ 0 — (virus added directly to MM) Virus spotted <5.0× 10⁴   >99.5 <5 × 10⁴ 99.5 On treatment (no virus (no virus CBP anddetected) detected) Immediately Eluted into MM Virus spotted 1.2 × 10⁷37 9.8 × 10⁶ 48 on untreated CBP and Immediately eluted into MM Virusspotted <5 × 10⁴ >97 <5 × 10⁴ >97 on treated (no virus (no virus CBP anddetected) detected) eluted onto MM after 10 Minutes Virus spotted 1.8 ×10⁶ 90 9.7 × 10⁶ 47.5 on untreated CBP and eluted into MM after 10minutes

Example 5 Method for Treating the Dry Solid Medium of the Invention foruse in Standard Analysis Systems

[0222] The dry solid medium of the invention, with or without componentsfor subsequent analysis can also be used in standard assays of analytesfor diagnostic purposes, e.g., in the Guthrie Test for phenylketonuria(PKU) and enzymic assays such as a test for galactosemia. Both of thesetests are routinely carried out on newborn infants. Prior to analysisusing such standard assay systems, it is preferable to neutralize theSDS and EDTA on the dry solid medium containing a sample of blood beforethe analysis of such analytes by adding a “converter solution.”

[0223] 5.1 Dry Solid Medium Converter Solution

[0224] The dry solid medium converter solution works by binding the SDSand EDTA.

[0225] A. Materials and Methods

[0226] The dry solid medium converter solution includes the followingmixture:

[0227] 1 . 577 mg Protamine sulphate (salmine) Sigma No-4380, lot71F-0037, is added to 7.69 ml water and shaken (to give 75 mg/ml).

[0228] This batch will be nominally 75 mg/ml before dialysis. Thisconcentration is near to the solubility of the protamine. It dissolvesslowly as a gluey turbid mass.

[0229] 2. Dialysis: this is a clean-up precaution to assure that thematerial is essentially sterile and fully dissolved. The gluey turbidmass is placed in a dialysis bag approximately 1.5 cm in diameter by 2cm to 10 cm in length. Both ends of the bag are knotted tightly. The bagis then weighed and dropped into a 1 liter flask containing about 800 mlof 50 mM of magnesium acetate with chloroform and a stirring bar. Thematerial is dialyzed in the flask, overnight, against the 800 ml of 50mM magnesium acetate with an excess (approximately 5 ml) of chloroformpresent as a sterilizing agent. The protamine almost completelydissolves overnight giving a clear solution and few obvious small lumpsof debris.

[0230] The weight of the bag increases:

[0231] Weight of bag before dialysis=8.1 g

[0232] 20 hour dialysis, 4° with CHCl₃ saturated fluid.

[0233] Weight of bag after dialysis=8.67 g.

[0234] Thus, the concentration of protamine is now, nominally, 70 mg/ml.(Assuming no losses through bag).

[0235] 3. The bag was then punctured at the bottom, and the fluidcollected and centrifuged at 4000 rpm (approximately 2000× g) for 10minutes to remove debris.

[0236] 4. 1 ml lots were stored at −80° C.

[0237] 5. Prior to analysis, about 3 μl of the converter solution isapplied to each 3 mm diameter disk of dry solid medium containing asample of blood.

[0238] 5.2 PKU Screening Procedure (Guthrie Test)

[0239] The Guthrie test is based on the stimulatory effect ofphenylalanine on the growth of Bacillus subtilis in the presence of β2-thienylalanine growth inhibitor. Disks of blood samples are placed onagar containing bacterial spores and the inhibitor. Following incubationat 37° C. for 16 to 18 hours, the diameter of the growth around the diskis proportional to the concentration of phenylalanine. Higher thannormal levels in infants is indicative of phenylketonuria.

[0240] An advantage of using the dry solid medium for the Guthrie testis that the disk of dry solid medium retains proteins and hemoglobinfrom the sample, leaving a cleaner area around the disk and making theinterpretation of the result easier. In fact, samples of bloodcontaining phenylalanine which were stored on the dry solid mediumconsistently gave at least as strong bacterial growth rings asequivalent samples on standard paper.

[0241] Also, because sufficient bacterial spores are used in a Guthrietest and the rate of growth is so fast, the inhibition or killing ofbacteria by reagents of the dry solid medium which were proximal to thedry solid medium did not cause a problem for interpreting the results.Therefore, when using the dry solid medium it is not necessary to applythe converter solution to the disk prior to the test. However, it is anadvantage to do this to remove an observable weak zone of growthinhibition.

[0242] 5.3 Screening for Galactosemia

[0243] Galactosemia screening requires the use of active enzymes toassay for galactose and galactose phosphate. The main enzyme,β-galactose dehydrogenase reduces NAD to NADH and this is observedspectrophotometrically.

[0244] This assay is severely inhibited by components of the compositionof the dry solid medium, but the inhibition can be substantiallyeliminated by the application of converter solution to a sample of bloodstored on the dry solid medium.

[0245] After addition of the converter solution, just prior to theenzymic assay, the assay has been observed to perform in the same way asit does with a sample of blood on untreated cellulose based paper. Ofcourse, the dry solid medium has the advantage of providing the benefitsof pathogen killing and preservation of the sample compared to standardmaterials used.

[0246] All patents and publications in the specification are indicativeof the level of ordinary skill in the art to which this inventionpertains. All patents and publications are herein incorporated byreference to the same extent as if each individual patent andpublication was specifically and individually indicated by reference.

Example 6 Polyester Material Swab Device for Obtaining, Storing, andAnalyzing Genetic Material Utilizing the Dry Solid Medium and Methodsthereof

[0247] A swab device utilizing a FTA coating on polyester (PET) materialcan be used to obtain, store and analyze various genetic materialsamples. Sampling of buccal areas and any other areas where sampleswould be swabbed can be performed with the PET material coated with FTA.Once the sample is obtained, then the sample can be subsequentlyanalyzed. Elution of the genetic material must be performed to removethe genetic material from the PET swab.

[0248] There are numerous embodiments of the swab device, but thepreferred embodiment employing the FTA-coated PET material has a narrowrod or stick-like support in combination with the FTA-coated PETmaterial, which is positioned adjacent to the support. The PET materialcan be a formed into a filter, a mesh or woven bulb, depending upon theapplication of the swab device. Additionally, the FTA-coated PETmaterial can be simply wrapped around a support such as a CEP swab.

[0249] The FTA coating can be applied directly to just the surface ofthe PET material or it can be applied in a manner that coats allindividual fibers. Any way the FTA coating is applied will providesimilar results. The method of using the swab device is provided indetail herewith.

[0250] 6.1. Materials and Methods:

[0251] 1. Scrape inside of cheek with the FTA coated PET swab.

[0252] 2. Air-dry the swab for thirty minutes.

[0253] 3. Cut head of swab in half using a pair of scissors that havebeen “flamed.”

[0254] 4. Add 1 mL of 0.5% SDS and ensure that the swab is covered andno air bubbles reside under the swab.

[0255] 5. Leave for five minutes at room temperature.

[0256] 6. Aspirate off the solution.

[0257] 7. Add 1 mL 0.5% SDS and ensure that the swab is covered and notair bubbles reside under the swab.

[0258] 8. Leave for five minutes at room temperature.

[0259] 9. Aspirate off the solution.

[0260] 10. Add 1 mL TE-1 and again ensure that the swab is completelycovered and no air bubbles lie under the swab.

[0261] 11. Leave for five minutes at room temperature.

[0262] 12. Aspirate off the solution.

[0263] 13. Add 1 mL TE-1 and again ensure that the swab is completelycovered and no air bubbles lie under the swab.

[0264] 14. Leave for five minutes at room temperature.

[0265] 15. Aspirate off the solution.

[0266] 16. Add 1 mL TE-1 and again ensure that the swab is completelycovered and no air bubbles lie under the swab.

[0267] 17. Leave for five minutes at room temperature.

[0268] 18. Aspirate off the solution.

[0269] 19. Apply 0.2 mL of sterile nuclease free water.

[0270] 20. Place in 95 C water bate for fifteen minutes.

[0271] 21. Spin at 12000 g for two minutes.

[0272] 22. Remove supernatant to a fresh, sterile eppendorf tube.

[0273] 6.2. Results and Discussion:

[0274] The yields for recovered DNA are given below and were assessedusing Oligreen. Run 1: ng/μl Run 2: ng/μl 1 0.6 0.6 2 1.9 1.6 3 1.4 1 40.7 0.7 5 1.8 2.4 6 1 1.5

[0275] Although the yields appear low, DNA was recovered. To ensure aprofile could be generated, 10 μl of each sample were employed in ourstandard multiplex reaction. These generated complete profiles. Theattached FIG. 5 shows an example of a complete profile.

[0276] A second batch of reactions was set up in single point PCR'samplifying a 1.3 kb fragment from the single locus B-globin. Thesereactions all employed 10 μl of the DNA solutions, so varying quantitiesof starting material were employed in a 20 μl reaction volume. FIG. 5shows the data generated.

[0277] The results of this series of extractions show that a swabgenerated from PET and FTA would be a good combination for both storingand isolating DNA from buccal scrapes. It would also be applicable tomany other areas where samples would be swabbed and genetic materiallater analyzed.

[0278] These series of experiments illustrate the utility of FTA coatingPET filter membrane for the collection and extraction of liquid DNA foramplification by PCR or multiplex profiling. An example of eluting FTAfilter membrane other than glass microfiber is shown.

[0279] The invention has been described in an illustrative manner, andit is to be understood that the terminology that has been used isintended to be in the nature of words of description rather than oflimitation.

[0280] Obviously, many modifications and variations of the presentinvention are possible in light of the above teachings. It is,therefore, to be understood that within the scope of the appendedclaims, the invention may be practiced otherwise than as specificallydescribed.

[0281] It will be apparent to one of ordinary skill in the art that manychanges and modifications can be made in the invention without departingfrom the spirit or scope of the appended claims.

REFERENCES

[0282] McCabe, E. R. B., et al., “DNA Microextraction From Blood Spotson Filter Paper Blotters: Potential Screening Applications to NewbornScreening,” Hum. Genet. 75:213-216 (1987)

1 8 1 23 DNA Artificial Sequence Description of Artificial SequencePrimer 1 tgactgagta caaactggtg gtg 23 2 22 DNA Artificial SequenceDescription of Artificial Sequence Primer 2 ctctatggtg ggatcatatt ca 223 27 DNA Artificial Sequence Description of Artificial Sequence Primer 3tgggctggaa tggaaaggaa tgcaaac 27 4 27 DNA Artificial SequenceDescription of Artificial Sequence Primer 4 tccattcgat tccatttttttcgagaa 27 5 27 DNA Artificial Sequence Description of ArtificialSequence Primer 5 gaatgtatta gaatgtaatg aacttta 27 6 27 DNA ArtificialSequence Description of Artificial Sequence Primer 6 ttccattccattccattcct ttccttt 27 7 26 DNA Artificial Sequence Description ofArtificial Sequence Primer 7 gtgctgcagg tgtaaacttg taccag 26 8 28 DNAArtificial Sequence Description of Artificial Sequence Primer 8cacggatccg gtagcagcgg tagagttg 28

What is claimed is:
 1. A swab device for obtaining and storing of asample of genetic material, wherein said swab device includes a drysolid medium comprising: a support and a head portion, said head portionconsisting essentially of a solid matrix for sorbing genetic materialthereon; and preserving means sorbed to said solid matrix for protectingthe genetic material, applied to said solid matrix, from degradation. 2.The swab device as set forth in claim 1, wherein said solid matrix isselected from the group consisting essentially of cellulose basedmaterial, hydrophillic polymers, synthetic hydrophilic polymers,polyester, polyamide, carbohydrate polymers, polytetrafluroethylene,fiberglass and porous ceramics.
 3. The swab device as set forth in claim1 further including releasing means for releasing the genetic materialfrom the solid matrix.
 4. The swab device as set forth in claim 3,wherein said releasing means is further defined including heat releasingmeans for releasing the genetic material from said solid matrix uponapplication of heat.
 5. A method of storing a sample of genetic materialonto a swab device by sorbing genetic material on a swab; and preservingand protecting the genetic material applied to the swab.
 6. The methodas set forth in claim 5, including the further step of releasing thesorbed genetic material from the solid swab.
 7. The method as set forthin claim 6, wherein said releasing step further includes application ofheat upon the solid swab.
 8. The method as set forth in claim 5, whereinthe swab includes a preserving means sorbed to the swab before thegenetic material is sorbed thereto.